N-acyloxylated cycloalkyl compounds, composition containing the same and methods of use therefor

ABSTRACT

Drugs or reagents containing as the active ingredient N-acyloxylated cycloalkyl compounds represented by general formula (I):                    
     wherein A is optionally substituted C 4  or C 5  cycloalkyl which may have one double bond in the ring; and R is C 1 -C 3  alkyl or phenyl). The above compounds are hydroxyamine derivatives functioning as spin trapping agents and can rapidly react with free radicals or active oxygen in an objective organ in spite of their being excellent in stability during the preparation or administration thereof.

TECHNICAL FIELD

The present invention relates to a drug comprising a modifiedN-acyloxylated cycloalkyl compound as an effective ingredient and, moreparticularly, to a drug comprising an N-acyloxylated cycloalkyl compoundwhich can scavenge in vivo active oxygen or free radicals and is usefulas an agent for preventing or curing various diseases induced by in vivoactive oxygen or free radicals and as a reagent for non-invasivelyacquiring biological images by a magnetic resonance method, typified bythe ESR (Electron Spin Resonance) method, or for detecting in vivoactive oxygen or free radicals in collected organisms.

BACKGROUND ART

Active oxygen is defined as one type of oxygen species with a short lifewhich is very reactive and takes part in various types of in vivooxidation reactions. The scope of active oxygen varies depending on thedefinition. In a narrow sense, active oxygen means a hydroxyl radical(.OH), superoxide (O₂ ⁻), singlet oxygen (¹O₂), and hydrogen peroxide(H₂O₂). In a broad sense, active oxygen includes a peroxy radical (LOO.)and alkoxy radical (LO.) which are derived from the reaction of theabove active species and biological components such as unsaturated fattyacid L, and a hypochlorite ion (ClO⁻) formed from H₂O₂ and Cl⁻ by thereaction with myeloperoxidase and the like.

Radicals are defined as atoms or molecules which possess one or moreunpaired electrons. A hydroxyl radical, superoxide, peroxy radical, andalkoxy radical are all radicals. Singlet oxygen and hydrogen peroxideare not radicals, but are formed from a radical reaction or cause otherradical reactions.

In recent years, active oxygen and free radicals showing various in vivobioactivity have attracted attention and have been studied in the fieldof biology, medicine, and pharmacology. The active oxygen or freeradicals are generated in vivo due to ultraviolet rays, radiation,atmospheric pollution, oxygen, metal ions, ischemia-reperfusion, and thelike. Active oxygen and free radicals thus generated cause various invivo reactions such as peroxidization of lipids, denaturation ofproteins, and decomposition of nucleic acids. Ischemic diseases,digestive diseases, cancer, cranial nervous diseases accompanied bynerve degeneration, inflammation, cataracts, and drug-inducedorganopathy are known as diseases accompanied by such phenomena.Noninvasive detection of such active oxygen and free radicals whichrelate to so many diseases may help in the investigation of the causesof a number of such diseases and provide useful medical information.

The following two methods are known as conventional methods fordetecting free radicals. One of these is an indirect method consistingof adding a reagent to a reaction system and detecting the resultingchanges in absorbance or emission of light by the reaction system. Theother method is an electron spin resonance (ESR) method consisting ofdirectly detecting unpaired electron of free radicals. Since the ESRmethod can measure both liquid and solid samples and even opaque ornon-uniform samples, this method is very advantageous for detectingactive oxygen in collected biological samples or in vivo.

The problem in detecting in vivo active oxygen or free radicals is thatESR cannot directly measure active oxygen or free radicals in a livingbody due to their short life. To solve this problem, a method ofindirectly observing in vivo active oxygen or free radicals byadministering a reagent to a living body and measuring the chemicalchanges in the reagent caused by active oxygen or free radicals usingESR has been employed. For this purpose, a spin trapping method has beendeveloped with an objective of measuring active oxygen having unpairedelectrons such as hydroxyl radicals. This method makes use of thecapability of a trapping agent to rapidly react with free radicalshaving only a short life and produce a spin adduct which is stable, hasa long life, and can be detected by ESR, as shown in the followingformula. In a narrow sense, the spin trapping agent has been defined asa compound having a double bond in the scavenging site, such as5,5-dimethyl-1-pyrroline-1-oxide (DMPO) shown below.

Specifically, measurement of short-life active oxygen becomes possibleby adding a compound which can rapidly react with radicals and producesa spin adduct sufficiently stable for measuring ESR to the measuringsystem as a spin trapping agent, and measuring the stable spin adduct.

Therefore, the requirements to be satisfied by the compound used as aspin trapping agent include: (1) capability of rapidly reacting withactive oxygen and free radicals, (2) being converted into sufficientlystable radicals, (3) being chemically stable when handled, and (4) beingfree from toxicity.

An attempt to directly detect or image in vivo active oxygen or freeradicals by using the above spin trapping agent has been undertaken.However, large volume biological samples cannot be measured usingconventional ESR devices which utilize microwaves of an X-band (about9.5 GHz) due to high dielectric loss in water.

In recent years, ESR-CT utilizing low-frequency microwaves (300-2000MHz) has been developed, making it possible to directly detect or imagefree radicals in a sample containing a large amount of water,particularly, free radicals in a living body.

The principle of a nuclear magnetic resonance (NMR) method wasdiscovered in 1945. In 1973, Lauterbur first applied the NMR method tomagnetic resonance imaging (MRI) which is an imaging device used inmedicine. Since then, the NMR method has progressed remarkably andbecomes one of the most universal diagnostic methods at present.

MRI first appeared as a diagnostic method using no contrast media. Atpresent, contrast media are used to increase the detectability of alesion site which is difficult to shade. Therefore, contrast mediaexhibiting superior detectability are demanded.

In recent years, the utility of nitroxide compounds as contrast mediafor MRI or ESR and the antioxidation effect thereof has attractedattention. For example, paramagnetic inorganic compounds such asgadolinium are administered as contrast media to contrast the lesionsite in the MRI diagnosis used in medicine. However, because of toxicityof such inorganic compounds, nitroxide compounds have been considered asMRI contrast media which can be used instead of gadolinium. As ESRimaging has been developed and the utility thereof has attractedattention, the utility value of nitroxide compounds as imaging agentshas increased. The possibility of utilization of nitroxide compounds asan active oxygen scavenging agent has also been suggested (see J. Biol.Chem. 263: 17921; 1998).

If information about active oxygen or free radicals in biological tissuecan be acquired as biological images by the noninvasive magneticresonance measuring method, this information can be used for studyingpathology in which active oxygen and free radicals take part, such asischemic diseases, digestive diseases, cancer, cranial nervous diseasesaccompanied by nerve degeneration, inflammation, cataracts, anddrug-induced organopathy (hereinafter referred to “diseases related toactive oxygen and the like”) and diagnosing these diseases.

In this situation, a report has been published describing thecharacteristics of some type of hydroxylamine derivative which caneasily react with free radicals and active oxygen by oxidativestimulation (active oxygen, etc.) and be converted into a nitroxidecompound having ESR signals (Biochem Biophys Res Commun 230, 54-57,1997). The compound is not a spin trapping agent in the stringent sensebecause this is not a generally defined nitron or nitroso compound.However, inasmuch as the capability of scavenging spins as shown by thefollowing formula, the compound has the same function as the spintrapping agent in a narrow sense.

In the above formula, A′ represents an alkylene group which may besubstituted.

Although it has been known that super oxide in solutions or cells can bedetected by measuring the ESR signals of the nitroxide compound formedby the above reaction, the hydroxylamine derivatives presented a seriousproblem in applying the above reaction to the detection of active oxygenand free radicals. Specifically, although nitroxide compounds derivedfrom hydroxylamine derivatives are such stable compounds that thesecompounds can be stored for several weeks in an aqueous solution andcrystals thereof can be stored for several years in a desiccator (see,for example, Arch. Biochem. Biophys. 215: 367-378; 1982), thehydroxylamine derivatives themselves are unstable and must be preparedeach time they are used.

For this reason, although a certain hydroxylamine derivative has beenused for the detection of free radicals or active oxygen in solutions orcells, there have been no examples of acquiring images of free radicalsand active oxygen generated in the organs by in vivo administration of ahydroxylamine derivative. The reason why this image acquisition has notbeen successful is considered to be because the in vivo reaction of thehydroxylamine derivative and free radicals is so fast that thehydroxylamine derivative is metabolized in blood before reaching theorgans.

Therefore, development of a technology using a hydroxyamine derivative,which functions as a spin trapping agent and can rapidly react with freeradicals or active oxygen in an objective organ and yet exhibitexcellent stability during preparation or administration, has beendesired.

DISCLOSURE OF THE INVENTION

In order to solve the above problems, the inventors of the presentinvention have conducted extensive studies to discover a compound whichis itself stable, rapidly reacts with active oxygen and free radicals inliving bodies producing stable products, and possesses guaranteed safetyin living bodies. As a result, the inventors have found that anN-acyloxy cycloalkyl compound obtained by acylating the hydroxyl groupof a certain hydroxylamine derivative satisfies the above requirements,can scavenge free radicals and active oxygen, and can be effectivelyused for the detection or deletion of such free radicals and activeoxygen.

Acquiring images of free radicals and active oxygen by spin trapping hasconventionally been considered to be difficult. However, since the aboveN-acyloxylated cycloalkyl compound is a compound produced by stabilizinga hydroxylamine derivative having the same function as a spin trappingagent, this compound is stable after preparation and can be transferredto the target organs without being metabolized after administration. Thecompound is then hydrolyzed into the hydroxylamine derivative, whichreacts with active oxygen or free radicals in the organ to produce anitroxide emitting ESR signals. The inventors have found that images ofactive oxygen or free radicals can be acquired by detecting the ESRsignals.

The inventors have further found that the N-acyloxylated cycloalkylcompound can scavenge active oxygen and free radicals, and can be usedas a preventive or therapeutic agent for diseases such as ischemicdiseases, digestive diseases, cancer, cranial nervous diseasesaccompanied by nerve degeneration, inflammation, cataracts, ordrug-induced organopathy, as a drug such as an image diagnosis agent ora detection reagent, and the like.

Accordingly an object of the present invention is to provide a drug orreagent containing an N-acyloxylated cycloalkyl compound shown by thefollowing formula (1) as an effective ingredient,

wherein A represents a C₄ or C₅ alkylene group which may have one doublebond in the ring and may be substituted with an alkyl group, aminogroup, amide group, carbamoyl group, carboxyl group, keto group,hydroxyl group, sulfonic acid group, phenyl group, acetoxyl group, oracetoamino group, and R is a C₁-C₃ alkyl group or phenyl group.

Another object of the present invention is to provide a method ofscavenging in vivo active oxygen or free radicals comprisingadministering the above N-acyloxylated cycloalkyl compound (I).

Still another object of the present invention is to provide a novelN-acyloxylated cycloalkyl compound represented by the following formula(II′),

wherein m is 0 or 1; when m is 0, X′ and Y′ individually represent ahydrogen atom, alkyl group, amino group, amide group, carbamoyl group,carboxyl group, keto group, hydroxyl group, sulfonic acid group, phenylgroup, acetoxyl group, or acetoamino group, and when m is 1, X′ and Y′individually represent a hydrogen atom, alkyl group, amino group, amidegroup, carbamoyl group, carboxyl group, keto group, hydroxyl group,sulfonic acid group, phenyl group, or acetoamino group; R is a C₁-C₃alkyl group or a phenyl group; R¹, R², R³, and R⁴ individually representa C₁-C₄ alkyl group; and

represents a single bond or double bond.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a calibration line used for the determination of theesterase concentration in a solution by the ESR signal strength using1-acetoxy-3-carbamoyl-2,2,5,5-tetramethylpyrrolidine.

FIG. 2 shows an ESR-CT image of the brain of a rat to which1-acetoxy-3-carbamoyl-2,2,5,5-tetramethylpyrrolidine has beenadministered and the positional relationship in the brain.

FIG. 3 is a drawing describing the part of the brain indicated by theanatomical chart shown in FIG. 2.

BEST MODE FOR CARRYING OUT THE INVENTION

The N-acyloxylated cycloalkyl compound (I) of the present invention canbe prepared by reducing the nitroxide compound shown by the formula(III) into a hydroxylamine compound shown by the formula (IV), andesterifying the hydroxylamine compound by the carboxylic acid shown bythe formula (V) or its reactive derivative according to the followingreaction:

wherein A represents a C₄ or C₅ alkylene group which may have one doublebond in the ring and may be substituted with an alkyl group, aminogroup, amide group, carbamoyl group, carboxyl group, keto group,hydroxyl group, sulfonic acid group, phenyl group, acetoxyl group, oracetoamino group, and R is a C₁-C₃ alkyl group or phenyl group.

In the above reaction, the nitroxide compound (III) used as the startingraw material is a known compound or a compound prepared by a knownmethod (for example, the method of A. M. Feldman et al. (U.S. Pat. No.3,334,103) or the method of W. Bueschken et al. (DP 4219459). Thefollowing compounds can be given as specific examples of the nitroxidecompound (III).

Although the reduction of the nitroxide compound (III) can be carriedout according to a conventional method, a method of dissolving thenitroxide compound (III) in methanol and reducing the compound by. theaddition of hydrazine monohydrate is preferably employed.

Esterification of the hydroxylamine compound (VI) obtained by thereduction of the nitroxide compound (III) can also be carried outaccording to a conventional method. One example of such a methodcomprises the reaction of the hydroxylamine compound (VI) with thecarboxylic acid (V) in the presence of a dehydration condensationcatalyst. A method of using a derivative of the carboxylic acid (V) suchas an active ester, acid anhydride, acid halide, and the like is alsoeffective.

As a preferable example of the above compound (I), N-acyloxylatedcycloalkyl compound represented by the following formula (II) can begiven.

wherein X and Y individually represent a hydrogen atom, alkyl group,amino group, amide group, carbamoyl group, carboxyl group, keto group,hydroxyl group, sulfonic acid group, phenyl group, acetoxyl group, oracetoamino group, and R is a C₁-C₃ alkyl group or a phenyl group, R¹,R², R³, and R⁴ individually represent a C₁-C₄ alkyl group, and

represents a single bond or double bond, and m indicates 0 or 1.

Among the N-acyloxylated cycloalkyl compounds represented by thefollowing formula (II), the compound shown by the following formula(II′) is a novel compound, not disclosed in any published document:

wherein m is 0 or 1; when m is 0, X′ and Y′ individually represent ahydrogen atom, alkyl group, amino group, amide group, carbamoyl group,carboxyl group, keto group, hydroxyl group, sulfonic acid group, phenylgroup, acetoxyl group, or acetoamino group, and when m is 1, X′ and Y′individually represent a hydrogen atom, alkyl group, amino group, amidegroup, carbamoyl group, carboxyl group, keto group, hydroxyl group,sulfonic acid group, phenyl group, or acetoamino group; R is a C₁-C₃alkyl group or a phenyl group; R¹′, R²′, R³, and R⁴ individuallyrepresent a C₁-C₄ alkyl group; and

represents a single bond or double bond, provided that when both X′ andY′ are a hydrogen atom, R is neither a phenyl group nor a methyl group,and when X′ is a hydrogen atom and Y′ is a hydroxyl group, R is not amethyl group.

Drugs or reagents for administration are prepared using theN-acyloxylated cycloalkyl compound (I) of the present invention thusobtained by dissolving the compound in a pharmaceutically or chemicallyacceptable solvent such as a physiological saline solution or isotonicphosphate buffer solution and adding optional components such aspropylene glycol or benzyl alcohol as required.

Drugs or reagents using the N-acyloxylated cycloalkyl compound (I) ofthe present invention are preferably prepared as injections, drops,liniments, eye drops, and the like.

Drugs using the N-acyloxylated cycloalkyl compound (I) of the presentinvention include diagnostic drugs. Such diagnostic drugs are used as adrug for diagnosing diseases relating to the active oxygen which detectthe presence of the active oxygen or free radicals by intravascularadministration. For example, such diagnostic drugs are used forcontrastradiography for MRI of brain or heart diseases orcontrastradiography for ESR.

Although the amount of the N-acyloxylated cycloalkyl compound (I) to beused in the above drugs differs depending on the object or objectiveorgans or diseases, such drugs are generally administered so that theamount of the N-acyloxylated cycloalkyl compound (I) is 0.1-500 mg/kg.

As examples of other usage for drugs, preventive preparations ortherapeutic agents for diseases caused by the active oxygen or freeradicals in vivo can be given. Such preventive or therapeutic agents,which react with active oxygen or free radicals and eliminate them, areeffective for prophylaxis and treatment of the diseases related toactive oxygen and the like.

The active oxygen or free radicals generated from the tissue or organsat a normal or diseased state can be detected from outside the body andimaged by administering the above drugs to normal animals and diseasedmodel experimental animals. The drugs can be used as detection reagentsfor determining what active oxygen and free radicals relate to what kindof diseases from the results of imaging, thereby providing usefulmedical information.

Furthermore, the drugs can be used as detection reagents for measuringthe presence or absence, or the amount of active oxygen or free radicalsin biological tissues by homogenizing collected samples, adding anappropriate buffer solution and the drugs to the homogenized solution,allowing the mixture to react for a certain period of time, andmeasuring the ESR.

The present invention will be described in more detail by way ofexamples, which should not be construed as limiting the presentinvention.

EXAMPLE 1 Synthesis of1-Acetoxy-3-carbamoyl-2,2,5,5-tetramethylpyrrolidine

(1) Synthesis of 1-Hydroxy-3-carbamoyl-2,2,5,5-tetramethylpyrrolidine

1.0 g (5.4 mmol) of 3-carbamoyl-2,2,5,5-tetramethyl-pyrrolidine-1-yloxywas dissolved in 50 ml of methanol. After the addition of 10 ml ofhydrazine monohydrate, the mixture was reacted for 6 hours at roomtemperature while stirring. The solvent was evaporated under vacuum toobtain 1.0 g (5.4 mmol, yield: 100%) of white crystals.

Melting point: 230-234° C. (decomposed) ¹H-NMR(in DMSO; δ): 0.88, s, 3H(CH₃), 1.02, s, 3H (CH₃), 1.07, s, 3H (CH₃), 1.14, s, 3H (CH₃), 1.52, dd(J=12.4 Hz, J=8.1 Hz), 1H (CH₂), 1.96, t (J=11.8 Hz), 1H (CH₂), 2.48, dd(J=11.8 Hz, J=8.7 Hz), 1H(CH), 6.81, s, 1H(CONH₂), 7.12, s, 1H(OH),7.15, s, 1H(CONH₂)

(2) Synthesis of 1-Acetoxy-3-carbamoyl-2,2,5,5-tetramethylpyrrolidine

20 ml of dichloromethane and 3 ml of triethylamine were added to 0.50 g(2.7 mmol) of 1-hydroxy-3-carbamoyl-2,2,5,5-tetramethylpyrrolidine. 0.38ml (4.0 mmol) of acetic anhydride was added dropwise to the mixture withstirring and ice-cooling, the mixture was stirred for 3 hours. Thereaction mixture was washed with water, 3% diluted hydrochloric acid,water, 5% sodium hydrogencarbonate aqueous solution, and water, in thisorder. The organic layer was dried over magnesium sulfate, and thesolvent was evaporated under vacuum. The residue was purified by silicagel column chromatography (ethyl acetate) and recrystallized from ethylacetate to obtain 0.53 g (2.3 mmol) of white crystals (yield: 86%).

Melting point: 150-151° C. ¹H-NMR (in DMSO; δ): 0.97, s, 3H (CH₃), 1.10,s, 3H (CH₃), 1.12, s, 3H (CH₃), 1.15, s, 3H (CH₃), 1.64, dd (J=12.4 Hz,J=7.4 Hz), 1H (CH₂), 2.06, s, 3H (COCH₃), 2.07, br, 1H (CH2), 2.62, br,1H (CH), 6.94, s, 1H (CONH₂), 7.27, s, 1H (CONH₂) Mass spectrum (EI⁺):m/z 228.3 (M⁺)

EXAMPLE 2 Synthesis of1-Propionyloxy-3-carbamoyl-2,2,5,5-tetramethylpyrrolidine

0.56 g (2.3 mmol) of white crystals were prepared in the same manner asin Example 1(2), except for using 0.52 ml of propionilc anhydrideinstead of 0.38 ml of acetic anhydride (yield: 85%).

Melting point: 116-117° C. ¹H-NMR(in DMSO; δ): 0.97, s, 3H(CH₃), 1.06, t(J=7.4 Hz), 3H(CH₃), 1.09, s, 3H (CH₃), 1.11, s, 3H (CH₃) 1.14, s, 3H(CH₃), 1.64, dd (J=12.4 Hz, J=7.4 Hz), 1H (CH₂) , 2.12, br, 1H(CH₂),2.36, q (J=7.4 Hz, 2H (CH₂), 2.62, br, 1H (CH), 6.94, s, 1H (CONH₂),7.27, s, 1H (CONH₂) Mass spectrum (EI⁺): m/z 242.3 (M⁺)

EXAMPLE 3 Synthesis of1-Butylyloxy-3-carbamoyl-2,2,5,5-tetramethylpyrrolidine

0.61 g (2.4 mmol) of white crystals were prepared in the same manner asin Example 1(2), except for using 0.66 ml of butyric anhydride insteadof 0.38 ml of acetic anhydride (yield: 88%).

Melting point: 103-104° C. ¹H-NMR (in DMSO; δ): 0.91, t (J=7.4 Hz), 3H(CH₃), 0.97, s, 3H (CH₃), 1.09, s, 3H (CH₃), 1.11, s, 3H (CH₃), 1.14, s,3H (CH₃), 1.58, sex (J=7.4 Hz), 2H (CH₂), 1.64, dd (J=12.4 Hz, J=7.4Hz), 1H (CH₂), 2.12, br, 1H (CH₂), 2.32, t (J=7.4 Hz), 2H (CH₂), 2.62,br, 1H (CH), 6.94, s, 1H (CONH₂), 7.27, s, 1H (CONH₂) Mass spectrum(EI⁺): m/z 256.3 (M⁺)

EXAMPLE 4 Synthesis of1-Benzoyloxy-3-carbamoyl-2,2,5,5-tetramethylpyrrolidine

0.72 g (2.5 mmol) of white crystals were prepared in the same manner asin Example 1(2), except for using 0.76 ml of benzoic anhydride insteadof 0.38 ml of acetic anhydride (yield: 92%).

Melting point: 199-201° C. ¹H-NMR (in DMSO; δ) 1.10, s, 3H (CH₃), 1.20,br, s, 12H (CH₃×3), 1.72, dd (J=12.4 Hz, J=7.4 Hz),1H (CH₂), 2.22, br,1H (CH₂), 2.72, br, 1H(CH), 6.99, s, 1H (CONH₂), 7.32, s, 1H (CONH₂),7.56, t (J=8.1 Hz), 2H (ARH), 7.68, t (J=7.4 Hz), 1H (ARH), 7.97, dd(J=8.1 Hz, J=1.2 Hz), 2H(ARH) Mass spectrum (EI⁺): m/z 290.4 (M⁺)

EXAMPLE 5 Synthesis of 1,4-Diacetoxy-2,2,6,6-tetramethylpiperidine

(1) Synthesis of 1,4-Dihydroxy-2,2,6,6-tetramethylpiperidine

1.0 g (5.8 mmol) of 4-hydroxy-2,2,6,6-tetramethyl-piperidine-1-yloxy wasdissolved in 50 ml of methanol. After the addition of 10 ml of hydrazinemonohydrate, the mixture was reacted for 6 hours at room temperaturewhile stirring. The solvent was evaporated under vacuum to obtain 1.0 g(5.8 mmol, yield: 100%) of white crystals.

Melting point: 167-168° C. (decomposed) ¹H-NMR (in DMSO; δ): 1.02, s, 6H(CH₃×2), 1.04, s, 6H (CH₃×2), 1.24, t (J=11.8 Hz), 2H (CH₂), 1.70, dd(J=11.8 Hz, J=3.1 Hz), 2H (CH₂), 3.74, m, 1H (CH), 4.38, d (J=5.0 Hz),1H (OH), 7.01, s, 1H (N—OH)

(2) Synthesis of 1,4-Diacetoxy-2,2,6,6-tetramethylpiperidine 9

20 ml of dichloromethane and 3 ml of triethylamine were added to 0.50 g(2.9 mmol) of 1,4-dihydroxy-2,2,6,6-tetra-methylpiperidine. 0.80 ml (2.9mmol) of acetic anhydride was added dropwise to the mixture withstirring and ice-cooling, the mixture was stirred for 3 hours. Thereaction mixture was washed with water, 3% diluted hydrochloric acid,water, 5% sodium hydrogencarbonate aqueous solution, and water, in thisorder. The organic layer was dried over. magnesium sulfate, and thesolvent was evaporated under vacuum. The residue was purified by silicagel column chromatography (ethyl acetate) to obtain 0.57 g (2.2 mmol) ofwhite crystals (yield: 76%).

Melting point: 72-73° C. ¹H-NMR (in DMSO; δ): 1.00, s, 6H (CH₃×2), 1.17,s, 6H (CH₃×2), 1.54, t (J=11.8 Hz), 2H (CH₂), 1.91, dd (J=11.8 Hz, J=3.1Hz), 2H (CH₂), 1.99, s, 3H (C—OCOCH₂), 2.06, s, 3H(N—OCOCH₃), 4.98, m,1H (CH) Mass spectrum (EI⁺): m/z 257.3 (M⁺)

EXAMPLE 6 Synthesis of1-Acetoxy-4-acetamide-2,2,6,6-tetramethylpiperidine

(1) Synthesis of 1-Hydroxy-4-amino-2,2,6,6-tetramethylpiperidine

1.0 g (5.8 mmol) of 4-amino-2,2,6,6-tetramethyl-piperidine-1-yloxy wasdissolved in 50 ml of methanol. After the addition of 10 ml of hydrazinemonohydrate, the mixture was reacted for 6 hours at room temperaturewhile stirring. The solvent was evaporated under vacuum to obtain 1.0 g(5.8 mmol, yield: 100%) of white crystals.

Melting point: 115-117° C. (decomposed) ¹H-NMR (in DMSO; δ): 1.00, s, 6H(CH₃×2), 1.01, s, 6H (CH₃×2), 1.10, t (J=11.8 Hz), 2H (CH₂), 1.59, d(J=9.9 Hz), 2H (CH₂), 2.84, m, 1H (CH), 7.05, s, 1H (N—OH)

(2) Synthesis of 1-Acetoxy-4-acetamide-2,2,6,6-tetramethylpiperidine

20 ml of dichloromethane and 3 ml of triethylamine were added to 0.50 g(2.9 mmol) of 1-hydroxy-4-amino-2,2,6,6-tetramethylpiperidine. 0.80 ml(8.4 mmol) of acetic anhydride was added dropwise to the mixture withstirring and ice-cooling, the mixture was stirred for 3 hours. Thereaction mixture was washed with water, 3% diluted hydrochloric acid,water, 5% sodium hydrogencarbonate aqueous solution, and water, in thisorder. The organic layer was dried over magnesium sulfate, and thesolvent was evaporated under vacuum. The residue was purified by silicagel column chromatography (ethyl acetate) and recrystallized from ethylacetate-hexane to obtain 0.54 g (2.1 mmol) of white crystals (yield:72%).

Melting point: 115-117° C. ¹H-NMR (in DMSO; δ) 0.96, s, 6H (CH₃×2),1.14, s, 6H (CH₃×2), 1.40, t(J=12.4 Hz), 2H (CH₂), 1.69, d (J=12.4 Hz),2H (CH₂), 1.78, s, 3H (C—OCOCH₂), 2.05, s, 3H (N—OCOCH₂), 3.97, m, 1H(CH), 7.74, d (J=8.1 Hz), 1H (NHCO) Mass spectrum (EI⁺): m/z 256.4 (M⁺)

EXAMPLE 7 Test for Measuring Enzyme Activity

Capability of enzyme (esterase) activity determination by ESR using1-acetoxy-3-carbamoyl-2,2,5,5-tetramethylpyrrolidine was examined.Esterase (3360 U/ml, manufactured by Sigma Co., Esterase, Porcine Liver)was diluted with a phosphate buffered saline solution (pH7.4, 67 mM) toconcentrations of 0.2, 2, 20, and 200 U/ml. 100 μl of each diluent wasadded to 100 μl of a 1 mM sample solution, and allowed to stand for oneminute. Specifically, the sample was converted into a hydroxylaminederivative using the esterase. Next, 10 μl of 10 mM sodium periodidephosphate buffered saline solution was added. Specifically, thehydroxylamine derivative produced was converted into a nitroxidederivative which can be measured by ESR. Finally, this solution issuctioned into a flat cell (manufactured by Labotech Co.) to measure ESRspectrum using “JES-REIX” manufactured by JEOL Ltd after one minute. Therelation between the signal strength of the ESR spectrum obtained andthe esterase concentration is shown in FIG. 1.

The result confirmed that the sample material is hydrolyzed by esterasein a short time and the reaction is quantitative. In addition, it wasconfirmed that the esterase activity can be measured by using thisreaction.

EXAMPLE 8 Measurement of ESR-CT Image of Rat Brain

6 ml of 150 mM physiological saline solution of1-acetoxy-3-carbamoyl-2,2,5,5-tetramethylpyrrolidine wasintraperitoneally administered to Wister male rats (200 g, age: nineweeks) anesthesized with pentobarbital. The head of the rat was securedso that the part of the head 9 mm ahead of the external auditory meatuscame to the center of the resonator. ESR-CT was measured 20 minutesafter administration. The measuring conditions of ESR-CT were asfollows.

(Measurement Conditions of ESR-CT)

Instrument: 700 MHz band electron spin resonance apparatus

Microwave frequency: 720 MHz

Microwave power: 52 mW

Central magnetic field: 25 mT

Magnetic field sweep width: 15 mT

Magnetic field modulation width: 0.2 mT

Magnetic field modulation frequency: 100 kHz

Magnetic field gradient: 1 mT/cm

Magnetic field gradient rotational angle: 20°

The measured black-and-white picture image is shown in FIG. 2, and acolor picture image is attached as a reference figure. In FIG. 2, A is arat brain ESR-CT image 1 mm posterior to the bregma, and B is a ratbrain ESR-CT image 3 mm posterior to the bregma. FIG. 3 is a drawingdescribing the part of the brain indicated by the anatomical chart inFIG. 2.

Nitroxide radical signals were observed in the hippocampus, cortex,striatum, amygdala, and hypothalamus of the brain, and the rat brainESR-CT images were acquired based on the signals. The experimentconfirmed that a modified hydroxylamine derivative is hydrolyzed afterbeing transferred to the brain, and oxidized by intracerebral activeoxygen and free radicals into a nitroxide derivative which emits ESRsignals, whereby images of free radicals or active oxygen can beacquired.

INDUSTRIAL APPLICABILITY

The N-acyloxylated cycloalkyl compound (modified hydroxylamine compound)of the present invention which are active ingredients of the diagnosticagent have enough half-life in blood and interact with active oxygen orfree radicals in vivo. Therefore, the nitroxide compounds are useful foracquiring biological images of the distribution of free radicals by amagnetic resonance method. Accordingly, the diagnostic agent can be usedfor diagnosing diseases related to active oxygen and the like such asischemic diseases, digestive diseases, cancer, cranial nervous diseasesaccompanied by nerve degeneration, inflammation, cataracts, ordrug-induced organopathy in which active oxygen or free radicals takepart.

Specifically, the above diseases related to active oxygen and the likecan be diagnosed by administering the diagnostic agent containing theN-acyloxylated cycloalkyl compound of the present invention to theliving body, and detecting the signal change of the nitroxide compoundsin vivo by ESR, NMR, and the like.

Therefore, the diagnostic agent of the present invention is used forMRI. If ESR devices capable of measuring large content biologicalsamples such as a human body are developed, the diagnostic agentnon-invasively diagnoses the diseases or symptoms in which active oxygentakes part by acquiring the images of free radical distribution in thehuman body by the ESR method.

Since the N-acyloxylated cycloalkyl compound of the present inventioncan react with in vivo active oxygen or free radicals and eliminatethem, the compound can be used as a preventive or therapeutic agent forthe diseases related to active oxygen and the like.

In addition, the active oxygen or free radicals generated from thetissue or organs in a normal or diseased state can be detected from theoutside of the body and imaged by administering the N-acyloxylatedcycloalkyl compound to normal experimental animals and diseased modelexperimental animals. From the results, the compound can be used asdetection reagents for determining what kind of diseases active oxygenand free radicals relate, whereby useful medical information isobtained.

Furthermore, the presence or absence or the amount of active oxygen orfree radicals in biological tissues can be measured by homogenizingcollected samples, adding an appropriate buffer solution and theN-acyloxylated cycloalkyl compound, and measuring the signal strength byESR after reacting the mixture for a certain period of time.

What is claimed is:
 1. A composition suitable for scavenging ordetecting active oxygen or free radicals comprising an N-acyloxylatedcycloalkyl compound of the following formula (I) and a carrier,

wherein A represents a C₄ or C₅ alkylene group, wherein A may containone ring double bond, and wherein A may be substituted with a groupselected from the group consisting of an alkyl group, amino group, amidegroup, carbamoyl group, carboxyl group, keto group, hydroxyl group,sulfonic acid group, phenyl group, acetoxyl group, and acetoamino group,and R is a C₁-C₃ alkyl group or phenyl group.
 2. The compositionaccording to claim 1, wherein A represents a C₄ alkylene group.
 3. Thecomposition according to claim 1, wherein A represents a C₅ alkylenegroup.
 4. The composition according to claim 1, wherein theN-acyloxylated cycloalkyl compound is a compound represented by thefollowing formula

wherein X and Y individually represent a hydrogen atom, alkyl group,amino group, amide group, carbamoyl group, carboxyl group, keto group,hydroxyl group, sulfonic acid group, phenyl group, acetoxyl group, oracetoamino group, R is a C₁-C₃ alkyl group or a phenyl group, R¹, R²,R³, and R⁴ individually represent a C₁-C₄ alkyl group,

represents a single bond or double bond, and m is 0 or
 1. 5. Thecomposition according to claim 4, wherein m is
 0. 6. The compositionaccording to claim 4, wherein m is
 1. 7. The composition according toclaim 4, wherein when m is 0, X and Y individually represent a hydrogenatom, alkyl group, amino group, amide group, carbamoyl group, carboxylgroup, keto group, hydroxyl group, sulfonic acid group, phenyl group,acetoxyl group, or acetoamino group, and when m is 1, X and Yindividually represent a hydrogen atom, alkyl group, amino group, amidegroup, carbamoyl group, carboxyl group, keto group, hydroxyl group,sulfonic acid group, phenyl group, or acetoamino group, R is a C₁-C₃alkyl group or a phenyl group, R¹, R², R³, and R⁴ individually representa C₁-C₄ alkyl group, and

represents a single bond or double bond, provided that when both X and Yare a hydrogen atom, R is neither a phenyl group nor a methyl group, andwhen X is a hydrogen atom and Y is a hydroxyl group, R is not a methylgroup.
 8. A method of scavenging or detecting active oxygen or freeradicals in vivo which comprises administering an N-acyloxylatedcycloalkyl compound shown by the following formula (I) in vivo,

wherein A represents a C₄ or C₅ alkylene group, wherein A may have onering double bond, and wherein A may be substituted with a substituentselected from the group consisting of an alkyl group, amino group, amidegroup, carbamoyl group, carboxyl group, keto group, hydroxyl group,sulfonic acid group, phenyl group, acetoxyl group, and acetoamino group,and R is a C₁-C₃ alkyl group or phenyl group.
 9. The method according toclaim 8, wherein A represents a C₄ alkylene group.
 10. The methodaccording to claim 8, wherein A represents a C₅ alkylene group.
 11. Themethod according to claim 8, wherein the N-acyloxylated cycloalkylcompound is a compound represented by the following formula (II),

wherein X and Y individually represent a hydrogen atom, alkyl group,amino group, amide group, carbamoyl group, carboxyl group, keto group,hydroxyl group, sulfonic acid group, phenyl group, acetoxyl group, oracetoamino group, R is a C₁-C₃ alkyl group or a phenyl group, R¹, R²,R³, and R⁴ individually represent a C₁-C₄ alkyl group,

represents a single bond or double bond, and m is 0 or
 1. 12. The methodaccording to claim 11, wherein m is
 0. 13. The method according to claim11, wherein m is
 1. 14. The method according to claim 11, wherein when mis 0, X and Y individually represent a hydrogen atom, alkyl group, aminogroup, amide group, carbamoyl group, carboxyl group, keto group,hydroxyl group, sulfonic acid group, phenyl group, acetoxyl group, oracetoamino group, and when m is 1, X and Y individually represent ahydrogen atom, alkyl group, amino group, amide group, carbamoyl group,carboxyl group, keto group, hydroxyl group, sulfonic acid group, phenylgroup, or acetoamino group, R is a C₁-C₃ alkyl group or a phenyl group,R¹, R², R³, and R⁴ individually represent a C₁-C₄ alkyl group, and

represents a single bond or double bond, provided that when both X and Yare a hydrogen atom, R is neither a phenyl group nor a methyl group, andwhen X is a hydrogen atom and Y is a hydroxyl group, R is not a methylgroup.
 15. An N-acyloxylated cycloalkyl compound represented by thefollowing formula (II′):

wherein m is 0 or 1, and when m is 0, X′ and Y′ individually represent ahydrogen atom, alkyl group, amino group, amide group, carbamoyl group,carboxyl group, keto group, hydroxyl group, sulfonic acid group, phenylgroup, acetoxyl group, or acetoamino group, and when m is 1, X′ and Y′individually represent a hydrogen atom, alkyl group, amino group, amidegroup, carbamoyl group, carboxyl group, keto group, hydroxyl group,sulfonic acid group, phenyl group, or acetoamino group, R is a C₁-C₃alkyl group or a phenyl group, R¹, R², R³, and R⁴ individually representa C₁-C₄ alkyl group, and

represents a single bond or double bond, provided that when both X′ andY′ are a hydrogen atom, R is neither a phenyl group nor a methyl group,and when X′ is a hydrogen atom and Y′ is a hydroxyl group, R is not amethyl group.
 16. The compound according to claim 15, wherein m is 0.17. The compound according to claim 15, wherein m is
 1. 18. The compoundaccording to claim 15, wherein

represents a single bond.
 19. The compound according to claim 18,wherein

represents a double bond.
 20. The compound according to claim 15,wherein X′ and Y′ are both a hydrogen atom, and R is neither a phenylgroup nor a methyl group.
 21. The method according to claim 8, whereinsaid detecting further comprises using contrasting ESR or NMR.
 22. Amethod of scavenging active oxygen or free radicals in a biologicaltissue or tissues, comprising: homogenizing a biological tissue ortissue to prepare a homogenized sample; and contacting said homogenizedsample with an N-acyloxylated cycloalkyl compound shown by the followingformula (I),

wherein A represents a C₄ or C₅ alkylene group, wherein A may have onering double bond, and wherein A may be substituted with a substituentselected from the group consisting of an alkyl group, amino group, amidegroup, carbamoyl group, carboxyl group, keto group, hydroxyl group,sulfonic acid group, phenyl group, acetoxyl group, and acetoamino group,and R is a C₁-C₃ alkyl group or phenyl group.
 23. The method accordingto claim 8, which comprises treating or diagnosing ischemic diseases,digestive diseases, cancer, cranial nervous diseases accompanied bynerve degeneration, inflammation, or drug-induced organopathy.